We studied mechanisms for T cell recognition of antigens in association with major histocompatibility complex (MHC)-encoded molecules, and applications to the design of synthetic vaccines for AIDS and cancer. We have been characterizing the helper and cytotoxic T lymphocyte (CTL) responses to HIV envelope and reverse transcriptase, mapping the key epitopes, and defining the role of individual residues in these epitopes to be able to modify the structures to make more potent immunogens as vaccines. We have made vaccine constructs in which clusters of helper epitopes are synthesized coupled to a peptide that is a CTL epitope presented promiscuously by multiple class I MHC molecules in the human and mouse as well as a neutralizing antibody epitope. These constructs can induce all three arms of the immune response, neutralizing antibodies, CTL, and Th1 helper cells. Results of the first arm of a phase I clinical trial with one of these peptides show ability to induce CTL, helper T cell responses, and neutralizing antibodies to HIV in at least a subset of human recipients. Currently, we are developing new approaches in mouse models to develop second generation vaccine constructs. We have shown proof of principle that we can modify the sequence of a helper epitope of HIV to make it more immunogenic and also much more potent, when coupled to a CTL epitope, in eliciting CTL and protecting against viral infection. The enhanced helper epitopes elicit a stronger Th1 response and upregulate CD40L on the helper cells, which in turn induce more IL-12 production by dendritic cells, which then polarize the T helper cells to Th1. We are applying this "epitope enhancement" approach to conserved HIV helper and CTL epitopes from env, gag, and pol, presented by human class II and class I HLA molecules, as well as to hepatitis C virus (HCV) epitopes and cancer antigen epitopes presented by human HLA-A2.1 (see below). We have developed an enhanced HIV reverse transcriptase epitope which is the subject of a clnical trial to be carried out with Dr. Robert Yarchoan, HAMB, CCR, NCI, and we have characterized human responses to an envelope helper epitope, which we have also enhanced. We have discovered ways of increasing CTL, helper, and antibody responses and steering them toward desired phenotypes, such as Th1 or Th2 or particular antibody isotypes, by incorporating cytokines into the emulsion adjuvant with the antigen. We compared a panel of 8 cytokines for their effects on 8 types of immune response, and discovered a novel synergy between GM-CSF and IL-12 and between TNF and IL-12 in induction of CTL. We found that all 3 cytokines provide triple synergy for induction of CTL with a peptide vaccine, for induction of interferon-gamma, and for protection against viral challenge in vivo, which we show to be interferon-gamma dependent. The mechanism of this synergy appears to relate to the upregulation of antigen presenting function by GM-CSF and of the IL-12 receptor by TNF and IL-12. Further, we have recently shown that GM-CSF that recruits dendritic cells and CD40L that matures them will synergize as a potent vaccine adjuvant combination for induction of CTL and protection against viral infection. We have even further enhanced this combination by a push-pull approach in which this potent combination of cytokine and costimulatory molecule is complemented by blocking a suppressive pathway with an inhibitor of IL-13, optimizing the vaccine-induced CTL response and protection. We have shown that high avidity CTL specific for HIV-1 envelope peptide are much more effective at clearing a recombinant vaccinia virus expressing HIV gp160 from SCID mice than are low avidity CTL specific for the same peptide-MHC complex, and have worked out two complementary mechanisms involving the ability of high avidity CTL to kill cells earlier in virus infection before viral progeny are produced, and to lyse targets more quickly. We have finally developed a method to preferentially elicit higher avidity CTL with a vaccine, by using costimulatory molecules to allow a response at lower antigen dose. However, we found that high avidity CTL are exquisitely sensitive to high dose antigen and will undergo programmed cell death, mediated by TNF and the TNF receptor II, but also requiring a permissive state involving a decrease in Bcl-2, IAP1, and TRAF2, and correlating with downmodulation of the T cell receptor. This effect may explain clonal exhaustion in viral infections. We have also found that IL-15 incorporated in a vaccine induces CTL of a different character that are longer-lived memory cells, with higher levels of IL-15Ra, greater responsiveness to IL-15 in vitro and greater homeostatic proliferation in vivo, and higher avidity for antigen. Most recently, we have shown that high avidity CTL express higher levels of IL-15 receptor alpha chain (IL-15Ralpha) and that this allows these cells to undergo more homeostatic proliferation in response to endogenous levels of IL-15 and survive, whereas low avidity CTL with lower levels of this receptor die out over time, providing an explanation for the long-standing enigma of T cell avidity maturation. We also found that IL-15 upregulates expression of the CD8 coreceptor. Thus, IL-15 mediates two mechanisms of T cell avidity maturation, selection at the population level and instruction at the single cell level. Use of this cytokine in vaccines should allow induction of longer-lived, higher avidity CTL that are more efficacious.We have shown for the first time that protection against mucosal transmission of virus can be mediated by CD8 CTL without antibodies, but requires that the CTL be present at the mucosal site of transmission, whereas systemic CTL are not sufficient. The protection can be accomplished by intrarectal immunization with a peptide vaccine and increased by inclusion of IL-12 and GM-CSF with the vaccine. We found that endogenous IL-12 is less inhibited by the mucosal adjuvant LT(R192G) than by cholera toxin, and substituting this, the mucosal CTL response and protection are less dependent on exogenous IL-12. Using this mutant LT, we immunized MamuA01-positive Rhesus macaques intrarectally with a similar peptide AIDS vaccine and induced CTL in the colon and mesenteric lymph nodes that have impacted the clearance of virus after intrarectal challenge with pathogenic AIDS virus SHIV-Ku. Intrarectal immunization was more effective than subcutaneous immunization with the same peptide vaccine at protecting against SHIV, in part because we found the induction of mucosal CTL provided for greater clearance from a major site of virus replication, the gut mucosa, which was seeding the bloodstream. We have just completed a second Rhesus macaque mucosal vaccine study using mucosal peptide priming, with a combination of cytokines as adjuvants, and using mucosal boosting with a recombinant poxvirus, to prevent AIDS virus transmission across a mucosal barrier. The peptide prime-NYVAC viral vector boost strategy appears more effective than either component alone in inducing both CTL responses and control of viremia after challenge. The group immunized by this strategy showed higher avidity CTL to several epitopes, and greater partial protection against intrarectal challenge with pathogenic SHIV-ku2. Peak viral load was delayed by two weeks, and viral load early after intrarectal challenge correlated with the CTL response before challenge. In addition, we have shown that a new method of transcutaneous immunization can induce CTL in the gut mucosa, providing a potentially more practical route of immunization to induce the needed mucosal immunity. With regard to cancer, we identified several CTL epitopes in proteins of hepatitis C virus (HCV), that causes liver cancer, using a novel approach, and have analyzed the role of each amino acid residue in order to modify one of the peptides to make a more potent vaccine.